Preliminary Study On The Mechanism Of Seizure Induced By FGF9 Knockout In Oligodendroglial Progenitor Cells Expressing Olig1 In Mice

Part I A novel epileptic mouse model was constructed:conditional knockout of FGF9 in oligodendrocyte progenitor cells expressing Olig1.Objective:A novel epileptic model mouse was established by conditional knockout of FGF9 in oligodendrocyte progenitor cells expressing Olig1 by Cre-Loxp recombinase system.Methods:?1?Conditional knockout of FGF9 in oligodendrocyte progenitor cells expressing Olig1 by Cre-Loxp recombinase system,and the mouse genotype was identified by PCR amplification.?2?The behavioral characteristics of mice were recorded and analyzed,and then the seizures of mice were further confirmed by electroencephalogram?EEG?.Results:?1?Using the Cre-Loxp recombinase system,Olig1-Cre transgenic mice were mated with FGF9^fl/fl transgenic mice to obtain FGF9^fl/+Olig1-Cre hybrid mice,which were then mated with FGF9^fl/fl mice to obtain FGF9^fl/flOlig1-Cre Homozygous mice.That is,FGF9 was conditional knocked out in oligodendrocyteprogenitorcellsexpressingOlig1(FGF9^fl/fll/fl Olig1-Cre,CKO^Olig1).The littermate control mice were FGF9^fl/fl?F/F?.The genotype and protein expression of CKO^Olig1 mice were verified by PCR and Western Blot respectively.?2?CKO^Olig1 mice were recorded and abnormal behavior of epileptic seizure was observed in knockout mice.The EEGs of CKO^Olig1 mice showed significant abnormal epileptic waves,while the control mice did not detect epileptic waves.The first epileptic seizure of CKO^Olig1 mice occurred 16-30days after birth.At the same time,the CKO^Olig1 mice were about one-third lighter in weight compared with the F/F mice,and there was significant difference between the two groups?**P<0.01?.Conclusions:FGF9 plays a key role in oligodendrocyte progenitor cells expressing Olig1.FGF9 in oligodendroglial progenitor cells expressing Olig1 was conditional knocked out to induce epileptic seizures in mice by the Cre-Loxp recombinase system,and a new epileptic model mice was established,which provides basic conditions for the study of epilepsy.Part II Study on the mechanism of epileptic seizures in model miceObjective:Study on the mechanism of epileptic seizures in knockout mice by ultra-high performance liquid chromatography-mass spectrometry ?UHPLC-MS?and RNA-seq Technology.Methods:?1?The concentrations of GABA and Glu in the cortex and hippocampus of mice were measured by UHPLC-MS,and the expression of neurotransmitters in the hippocampus of mice was observed by immunofluorescence staining.?2?The number of epileptic seizures in mice before and after intraperitoneal injection of gabapentin was observed by video video.?3?The differentially expressed genes?DEGs?between CKO^Olig1 mice and F/F mice were studied by RNA-seq technology,and the obtained DEGs were verified by qRT-PCR technology.Results:?1?The concentrations of GABA and Glu in the cortex and hippocampus of mice were measured by UHPLC-MS.Compared with the control group,we found that the GABA concentration of CKO^Olig1 mice decreased significantly,and the difference was statistically significant?*P<0.05?.Immunofluorescence staining showed that the expression of GABA and VGAT in hippocampal CA3 region of CKO^Olig1 mice was significantly decreased,while there was no significant difference in VGLUT1 expression.?2?After intraperitoneal injection of gabapentin into CKO^Olig1 mice,the number of seizures was significantly reduced,and the difference was statistically significant?**P<0.01?.?3?RNA-seq technology was used to analyze the gene expression profile in the hippocampus of CKO^Olig1 and F/F mice.A total of 150 DEGs were selected and seven DEGs were selected for qRT-PCR verification.It was found that the change trend of most genes was consistent with the RNA-seq results.Among them,G protein/adenylate cyclase?Adcy,AC?/cAMP signaling pathway plays a role in seizures in CKO^Olig1 mice.Conclusion:FGF9 loss-of function in GABAergic neurons blocked GABA synthesis by activation of the G protein/AC/cAMP signaling pathway,causing an imbalance of GABA and Glu in the hippocampus and induces epilepsy in mice.In summary,FGF9 plays a critical role for maintaining the physiological level of GABA and appears to be a new candidate gene for epileptogenesis.